Melanoma is a lethal malignancy that is poorly responsive to conventional treatments, but it is potentially an immunoresponsive disease. Several approaches to melanoma immunotherapy have demonstrated the feasibility of eliciting RT cell responses to melanoma- associated antigens. In this proposal we focus on the use of dendritic cells (DC) to induce anti-tumor immunity in patients with advanced melanoma. Because of the manner in which they process protein, express co-stimulatory molecules, and elaborate cytokines that support the development of cytolytic T lymphocyte (CTL) responses, DC are uniquely capable of antigen presentation. They appear to be ideal vehicles for delivery of antigen in tumor vaccination strategies. However, the optimum strategy for antigen delivery has not been determined. Our laboratories have in parallel studied two methodologies for antigen delivery: peptide pulsing of DC with melanoma antigen-derived peptide epitopes, and production of cell fusions between DC and tumor cells. We carried out pre-clinical studies on DC production and function. In doing so, we demonstrated our ability to reliably produce DC in high yields by culturing PBMCs obtained via leukapheresis. Phenotypic and functional analyses showed that these cells express a profile of cell-surface markers characteristic of DC. Concurrently we utilized DC to verify that specific substitutions of the gp100 280 epitope result in peptides that bind to HLA-A2 with higher affinity than the native epitope. Based on these studies, w4e are carrying out a phase I, dose escalation trial of peptide- pulsed DC therapy. To date we have shown that this approach is safe. Immunogenicity of vaccination has been documented by the production of CTL specific for the immunizing antigen, and clinical responses have been observed. We also studied the production of DC/tumor fusions. Pre- clinical data demonstrated our ability to generate heterokaryons from DC and breast, ovary, renal cell and melanoma tumors. Such hybridomas express both DC-derived co-stimulatory molecules and tumor-associated antigens and reproducibly stimulate the production of anti-tumor T cell immunity in vitro. We are conducting a phase I trial and preliminary have demonstrated that clinical administration of DC/tumor fusion is safe. DC/fusions also elicit T cell anti-tumor immunity, documented by enhanced interferon secretion in response to stimulation by tumor. Based on these pre-clinical and phase I clinical data, we propose to translate these findings into a phase II, randomized clinical study. Patients will be recruited to both approaches using uniform eligibility criteria at our four institutions. The primary endpoint of the study is the comparison of immunologic data obtained from the approaches.